1 Quali-quantitative analysis of flavonoids of Cornus mas L.

(Cornaceae)

2 fruits

4 Agata Maria Pawlowska a, Fabiano Camangi b, Alessandra Braca a,*

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a
6 Dipartimento di Scienze Farmaceutiche, Università di Pisa, via Bonanno 33, 56126

7 Pisa, Italy
b
8 Scuola Superiore S. Anna di Studi Universitari e di Perfezionamento di Pisa, Piazza

9 Martiri della Libertà 33, 56127 Pisa, Italy

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19 * Corresponding author. Tel.: +39-050-2219688, fax: +39-050-221660; e-mail:

20 braca@farm.unipi.it. (A. Braca)

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21 Abstract

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23 The methanol extract obtained from the ripe fruits of Cornus mas L. (Cornaceae) have

24 been phytochemically studied. On the basis of HPLC-PDA-MS/MSn analyses eight

25 compounds have been recognized as quercetin, kaempferol, and aromadendrin

26 glycosilated derivatives. Three major compounds have been also isolated by Sephadex

27 LH-20 column chromatography followed by HPLC and characterized by NMR

28 spectroscopy. Moreover, LC-PDA-MS analyses of the red pigment revealed the presence

29 of three anthocyanins. The quantitative analysis of all compounds was reported.

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31 Keywords: Cornus mas, flavonoids, anthocyanins, HPLC, ESI-MS/MS

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34 1. Introduction

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36 Cornus mas L. (Cornaceae family) known as the European and Asiatic cornelian cherry

37 is a species of dogwood native to Southern Europe and Southwest Asia. It is a medium to

38 large deciduous shrub or small tree growing to 5-12 m tall, with dark brown branches and

39 greenish twigs. The fruit is an oblong, red drupe 2 cm long and 1.5 cm in diameter,

40 containing a single seed. The fruit is edible, but the unripe is astringent. The most

41 common use of cornelian cherry fruits, is to produce different drinks, sweets, gels, and

42 jams (Millspaugh, 1974). Extract from the fruits is also used in Europe for cosmetic

43 purposes, replacing synthetic astringent substances, and are claimed to exert a favorable

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44 action on the human complexion (Polinicencu, Popesci, & Nistor, 1980). As ornamental,

45 cornelian cherry, with its brilliant leaves and abundant, attractive flowers, is employed

46 with very interesting effect in parks and small gardens (Slimestad & Andersen, 1998).

47 Fruits and vegetables are a good source of natural antioxidants, which containing many

48 different radical scavenger components provide protection against harmful-free radicals

49 and so are associated with lower incidence and mortality rates of cancer and heart

50 diseases in addition to a number of other health benefits (Wang, Cao, & Prior, 1996; Shui

51 & Leong, 2006). Among natural compounds, phenolics and in particular flavonoids were

52 found to be an important part of human diet and are considered as active principles in

53 many medicinal plants. In addition, flavonoids with their subclass anthocyanins,

54 contribute the yellow, orange, red, and blue color to flowers, fruits, and vegetables

55 (Cooper-Driver, 2001), and could become important in the replacing of the synthetic

56 pigments by the natural ones.

57 There are some investigations regarding the physical and chemical properties of

58 cornelian cherry fruits, their antioxidant capacity, phenol, ascorbic acid, as well as

59 anthocyanin contents (Tural & Koca, 2008; Vareed, Reddy, Schutzki, & Nair, 2006;

60 Marinova, Ribarova, & Atanassova, 2005; Klimenko, 2004; Demir & Kalyoncu, 2003;

61 Seeram, Schutzki, Chandra, & Nair, 2002; Didin, Kızılaslan, & Fenercioğlu, 2000;

62 Guleryuz, Bolat, & Pirlak, 1998). Two old studies revealed that the berries of C. mas

63 contain five anthocyanins, identified by paper chromatography, spectrophotometric, and

64 peroxide oxidation analyses as delphinidin 3-galactoside, cyanidin 3-galactoside,

65 cyanidin 3-rhamnosylgalactoside, pelargonidin 3-galactoside, and pelargonidin 3-

66 rhamnosylgalactoside (Du & Francis, 1973a; Du & Francis, 1973b). The later work

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67 (Seeram et al., 2002) showed that anthocyanins of cornelian cherry are the mixture of

68 three compounds: delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, and

69 pelargonidin 3-O-galactoside. The last data (Tural & Koca, 2008) indicated as three main

70 C. mas anthocyanins cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, and pelargonidin

71 3-O-glucoside. However, the survey of the literature revealed that the flavonoids content

72 of the fruits has never been reported.

73 The objectives of this study were to identify individual flavonoids of the fruits, evaluate

74 their profile, and compare the content of anthocyanins, in order to contribute to the

75 knowledge the potential value of these fruits as food.

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77 2. Materials and methods

78

79 2.1. Plant material

80 Cornus mas L. fruits were collected in Riparbella (PI), Italy, in September 2006. The

81 specimen was further identified and authenticated by Dr. Fabiano Camangi, Scuola

82 Superiore S. Anna di Studi Universitari e di Perfezionamento di Pisa, Pisa, Italy.

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84 2.2. Reagents

85 Standard of quercetin 3-O-rutinoside was purchased from Merck (E. Merck, Darmstadt,

86 Germany). Standards of cyanidin 3-O-galactoside and pelargonidin 3-O-glucoside were

87 obtained from Extrasynthese (Extrasynthese, France). Standard of pelargonidin 3-O-

88 rutinoside was kindly provided by Indena, Italy. HPLC grade acetonitrile (CH3CN),

89 formic (HCOOH) and acetic (CH3COOH) acids were purchased from J.T. Baker (Baker

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 90 Mallinckrodt, Phillisburg, NJ, USA). HPLC grade water (18 mΩ) was prepared by a

91 Mill-Ω50 purification system (Millipore Corp., Beddford, MA, USA).

92

93 2.3. General methods

94 An Avance Bruker 250 NMR spectrometer was used for NMR experiments; chemical

95 shifts are expressed in δ (parts per million) referenced to the solvents peaks δH 3.34 and

96 δC 49.0 for CD3OD. Column chromatography was performed over Sephadex LH-20

97 (Pharmacia, Uppsala, Sweden); HPLC separations were conducted on a Shimadzu LC-8A

98 (Shimadzu Corp., Kyoto, Japan) series pumping system equipped with a Waters R401

99 refractive index detector and a Shimadzu injector with a Waters µ-Bondapak C18 column

100 (7.8 × 300 mm, 10 µm, Waters, Milford, MA, USA). Thin-layer chromatography (TLC)

101 was performed on precoated Kieselgel 60 F254 plates (E. Merck, Darmstad, Germany).

102 HPLC-PDA-ESI-MS analyses were performed using a Surveyor LC pump, a Surveyor

103 autosampler, coupled with a Surveyor PDA detector, and a LCQ Advantage ion trap mass

104 spectrometer (Thermo Finnigan, San Jose, CA) equipped with Xcalibur 3.1 software.

105 Flavonoid analyses were performed using a 4.6 × 250 mm, 5.0 µm, X Terra C18 column

106 (Waters, Milford, MA) and the eluent was a mixture of 0.1% acetonitrile solution of

107 CH3COOH (solvent A) and 0.1% aqueous solution of CH3COOH (solvent B). The

108 solvent gradient was as follows: 0-50 min, 8-45% (A). Elution was performed at a flow

109 rate of 1.0 ml/min with a splitting system of 2:8 to MS detector (200 µl/min) and PDA

110 detector (800 µl/min), respectively. Analyses were performed with an ESI interface in the

111 negative mode. The ionization conditions were optimized, and the parameters were as

112 follows: capillary temperature, 260 °C; capillary voltage, 18 V; tube lens offset, 5 V;

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113 sheath gas flow rate, 60 arbitrary units; auxiliary gas flow rate, 6 arbitrary units; spray

114 voltage, 4.5 kV; scan range of m/z 200-700. PDA data were recorded with 220-500 nm

115 range with the preferential channels 254 and 324 nm as the detection wavelengths.

116 Anthocyanin analyses were performed using a 4.6 × 250 mm, 4 µm, Synergi Polar-RP 80

117 A column (Phenomenex Corp., Torrance, CA) and the eluent was a mixture of 1%

118 acetonitrile solution of HCOOH (solvent A) and 1% aqueous solution of HCOOH

119 (solvent B). The solvent gradient was as follows 0-20 min, 8-25% (A). Elution was

120 performed at a flow rate of 1 ml/min with the splitting system of 2:8 to MS detector,

121 respectively. Analyses were conducted with an ESI interface in the positive mode.

122 Cyanidin 3-O-galactoside was used to optimize the ioinization and the fragmentation

123 conditions; parameters were as follows: capillary temperature, 280 °C; capillary voltage,

124 11 V; tube lens offset, 15 V; sheath gas flow rate, 60 arbitrary units; auxiliary gas flow

125 rate, 6 arbitrary units; spray voltage, 3.5 kV; scan range of m/z 200-700. PDA data were

126 recorded with 220-600 nm range with a preferential channel of 515 nm as the detection

127 wavelength. In both cases N2 was used as the sheath and auxiliary gas. The volumes of

128 injections were 20 µl. HPLC-PDA quantitative analyses were performed using a Waters

129 600E multisolvent delivery system, a Waters 717plus autosampler, and a Waters 996

130 PDA detector (Waters, Milford, MA) equipped with Millenium32 Chromatography

131 Manager Software. The experimental conditions (solvent gradient, PDA channels, and

132 columns) were the same as described above. The volume of injection was 25 µl.

133 Quantitative determination was carried using calibration curves of standards. Quercetin

134 3-O-rutinoside, aromadendrin 7-O-glucoside, and cyanidin 3-O-galactoside were selected

135 as the external standards of calibration for flavonols, dihydroflavonol, and anthocyanins,

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136 respectively. Standard calibration curves were prepared in a concentration range 0.0005-

137 0.05 mg/ml with five different concentration levels (0.0005, 0.001, 0.005, 0.01, and 0.05

138 mg/ml) for quercetin 3-O-rutinoside and in a range of 0.001-0.05 mg/ml with four

139 concentration levels (0.001, 0.005, 0.01, and 0.05 mg/ml) for aromadendrin 7-O-

140 glucoside and cyanidin 3-O-galactoside. Triplicate injections were made for each level,

141 and a weight linear regression was generated. The calibration curves with the external

142 standards were obtained using concentration (mg/ml) with respect to the area obtained

143 from the integration of the PDA peaks at a wavelength of 254 nm for flavonoids, 285 nm

144 for dihydroflavonol, and 515 nm for anthocyanins. The relation between variables was

145 analyzed using linear simple correlation. For the linear regression of the external

146 standard, R2 was 0.9993 for quercetin 3-O-rutinoside, 0.9996 for aromadendrin 7-O-

147 glucoside, and 0.9998 for cyanidin 3-O-galactoside, respectively. For the quantification

148 of the compounds, a GraphPad Software Prism 3.0 was used. The amount of the

149 compound was finally expressed in mg/10 g of fresh fruits.

150

151 2.4. Extraction, isolation, and identification of flavonoids

152 Lyophilized fruits of C. mas (280 g) were defatted at room temperature with n-hexane,

153 and extracted with MeOH by exhaustive maceration (5 × 500 ml) to yield 178 g of

154 residue, which was dissolved in water and partitioned firstly with EtOAc and then with n-

155 BuOH. The dried n-butanol extract (5 g) was subjected to fractionation on a Sephadex

156 LH-20 column, using MeOH as eluent at a flow rate of 0.8 ml/min. Fractions of 8 ml

157 were collected and grouped into ten (A-J) fractions by TLC analyses on silica 60 F254 gel-

158 coated glass sheets developed with n-BuOH-AcOH-H2O (60:15:25) as the eluent.

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159 Fractions G (55.9 mg), H (36.1 mg), I (21.3 mg), and J (31.4 mg) were separately

160 purified by RP-HPLC on a 7.8 x 300 mm i.d., C18 µ-Bondapak column at a flow rate of

161 2.0 ml/min with MeOH-H2O (45:55) for fraction G, MeOH-H2O (35:65) for fraction H,

162 MeOH-H2O (3:7) for fraction I, and MeOH-H2O (4:6) for fraction J, to afford compounds

163 1 (6.1 mg, tR = 12 min) and 7 (2.5 mg, tR = 30 min) from fraction H, 4 (12.0 mg, tR = 20

164 min) from fraction G, and 7 from fraction I (1.5 mg, tR = 16 min) and J (3.3 mg, tR = 9

165 min), respectively. Compounds were identified by spectroscopic methods and HPLC-

166 PDA-ESI-MS analysis with authentic standards (Fig. 1):

167 Aromadendrin 7-O-β-D-glucoside (1): yellow amorphous powder. Negative ESI-MS:

168 m/z 449 [M-H]-. 1H and 13

C data are consistent with previously published data

169 (Slimestad, Anderson, & Francis, 1994).

170 Quercetin 3-O-β-D-xyloside (2): ESI-MS: m/z 433 [M-H]-. The compound was

171 identified by HPLC-PDA-ESI-MS analysis (retention time, UV spectrum, and ESI-MS

172 spectrometric data) and comparison with authentic standard.

173 Quercetin 3-O-a-L-rhamnoside (3): ESI-MS: m/z 447 [M-H]-. The compound was

174 identified by HPLC-PDA-ESI-MS analysis (retention time, UV spectrum, and ESI-MS

175 spectrometric data) and comparison with authentic standard.

176 Quercetin 3-O-rutinoside (4): yellow amorphous powder. Negative ESI-MS: m/z 609

177 [M-H]-. 1H and 13

C data are consistent with previously published data (Rastrelli,

178 Saturnino, Schettino, & Dini, 1995).

179 Quercetin 3-O-β-D-galactoside (5): ESI-MS: m/z 463 [M-H]-. The compound was

180 identified by HPLC-PDA-ESI-MS analysis (retention time, UV spectrum, and ESI-MS

181 spectrometric data) and comparison with authentic standard.

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182 Quercetin 3-O-β-D-glucoside (6): ESI-MS: m/z 463 [M-H]-. The compound was

183 identified by HPLC-PDA-ESI-MS analysis (retention time, UV spectrum, and ESI-MS

184 spectrometric data) with authentic standard.

185 Quercetin 3-O-β-D-glucuronide (7): yellow amorphous powder. ESI-MS: m/z 477 [M-

186 H]-. 1H and 13

C data are consistent with previously published data (Moon, Tsushida,

187 Nakahara, & Terao, 2001).

188 Kaempferol 3-O-β-D-galactoside (8): ESI-MS: m/z 447 [M-H]-. The compound was

189 identified by HPLC-PDA-ESI-MS analysis (retention time, UV spectrum, and ESI-MS

190 spectrometric data) and comparison with authentic standard.

191

192 2.5. Extraction and identification of anthocyanins

193 Fresh fruits (10 g) of C. mas were homogenized in 60 ml of 2% HCl methanol solution.

194 The solution was filtered on Büchner funnel and the filtrate was used for HPLC analyses.

195 Identifications were made by comparison of MS, PDA/UV, and retention data recorded

196 for standard anthocyanins.

197

198 3. Results and discussion

199

200 The methanol extract of the fruits of C. mas was partitioned with n-hexane, EtOAc, and

201 n-BuOH. The butanol extract was subjected to fractionation with an initial separation by

202 Sephadex LH-20 column chromatography. Subsequent purification of the fractions by

203 semipreparative HPLC led to the isolation of three compounds: aromadendrin 7-O-β-D-

204 glucoside (1), quercetin 3-O-rutinoside (4), and quercetin 3-O-β-D-glucuronide (7) (Fig.

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205 1). The structure of the isolated compounds was established by 1H and 13C NMR data and

206 confirmed by comparison with those reported in the literature. The presence of not

207 isolated compounds, quercetin 3-O-β-D-xyloside (2), quercetin 3-O-α-L-rahmnoside (3),

208 quercetin 3-O-β-D-galactoside (5), quercetin 3-O-β-D-glucoside (6), and kaempferol 3-O-

209 β-D-galactoside (8), was revealed by evaluation of the flavonoid profile throughout

210 HPLC-PDA-ESI-MS analyses. The components were identified by comparison of

211 obtained data (retention times, UV spectra, MS spectrometric data) with those of

212 authentic standards. The LC-MS Base Peak chromatogram of the methanol extract and

213 the chromatographic, spectroscopic, and spectrometric data, as well as, the quantitative

214 amounts of individual compounds are shown in Fig. 2 and Table 1, respectively. Results

215 obtained from quantitative analyses demonstrated the flavonoids content of 221.3 mg/10

216 of fruits. Methanol extract of cornelian cherries presented rich flavonoid glycosides

217 composition, that included eight compounds. Except for compound 1, which belongs to

218 the dihydroflavonols, they were all O-flavonol glycosides with quercetin and kaempferol

219 as aglycones and oligosaccharide moieties as mono- or disaccharides linked at the 3-OH

220 position. Quercetin 3-O-β-D-glucuronide (7) was the major constituent (69.9 mg),

221 followed by kaempferol 3-O-β-D-galactoside (8) (41.3 mg).

222 The anthocyanin profile of cornelian cherry berries was carried out by means of HPLC-

223 PDA-ESI-MS analyses. The chromatogram of the anthocyanins extract, recorded at 515

224 nm, is shown in Fig. 3. All the compounds were identified by comparison of their HPLC

225 retention times, elution orders, ESI-MS spectrometric data, and photodiode array

226 PDA/UV-vis with anthocyanin standards (Table 2). Compound 9 was recognized as

227 cyanidin 3-O-galactoside, 10 as pelargonidin 3-O-glucoside, and 11 as pelargonidin 3-O-

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228 rutinoside, respectively (Fig. 1). Our results of the anthocyanin qualitative composition

229 were not completely in agreement with those previously mentioned (Du & Francis,

230 1973a; Du & Francis, 1973b; Seeram et al., 2002; Tural & Koca, 2008). The total amount

231 of anthocyanins (Table 2) in C. mas fruits, determined on the cyanidin 3-O-galactoside

232 basis, was 11.7 mg/10 g of fresh fruits. This result is quite comparable with those

233 reported by Tural et al. (2008) and Pantelidis et al. (2007). Pelargonidin 3-O-glucoside

234 (10) was the predominant anthocyanin, followed by cyanidin 3-O-galactoside (9).

235 Pelargonidin 3-O-rutinoside was the least abundant one and present only in trace. The

236 differences in the composition of the fruits, could depend on the growing conditions, such

237 as soil, geographical and environmental conditions during the fruit development, degree

238 of maturity at harvested, and/or genetic differences.

239 Considering, that the epidemiological and experimental studies are correct in

240 suggesting that higher intake of phenolics from food are associated with reduced risk of

241 cancer, heart disease, and stroke, the immediate challenge is how to increase the level of

242 these beneficial phytochemicals in major food plants and find their new sources. The

243 fruits of cornelian cherry revealed the presence of considerable amounts of flavonoids.

244 Thus, results of the present study supported the antioxidant and nutraceutical potential of

245 this plant species.

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246 References

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